PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “replication fork stability”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed2024

PARP1 UFMylation ensures the stability of stalled replication forks.

The S-phase checkpoint involving CHK1 is essential for fork stability in response to fork stalling. PARP1 acts as a sensor of replication stress and is required for CHK1 activation. However, it is unclear how the activity of PARP1 is regulated. Here, we found that UFMylation is required for the efficient activation of CHK1 by UFMylating PARP1 at K548 during replication stress. Inactivation of UFL1, the E3 enzyme essential for UFMylation, delayed CHK1 activation and inhibits nascent DNA degradation during replication blockage as seen in PARP1-deficient cells. An in vitro study indicated that PARP1 is UFMylated at K548, which enhances its catalytic activity. Correspondingly, a PARP1 UFMylation-deficient mutant (K548R) and pathogenic mutant (F553L) compromised CHK1 activation, the restart of stalled replication forks following replication blockage, and chromosome stability. Defective PARP1 UFMylation also resulted in excessive nascent DNA degradation at stalled replication forks. Finally, we observed that PARP1 UFMylation-deficient knock-in mice exhibited increased sensitivity to replication stress caused by anticancer treatments. Thus, we demonstrate that PARP1 UFMylation promotes CHK1 activation and replication fork stability during replication stress, thus safeguarding genome integrity.

DNA Replication↗
PubMed2025

Pathogenic variants in MAEA disrupt DNA replication fork stability and are associated with developmental abnormalities in humans.

Replication stress (RS) poses a threat to genome stability and drives genomic rearrangements. The homologous recombination (HR) pathway repairs stalled replication forks (RFs) and prevents such instability. Through an E3 ubiquitin ligase screen aimed at identifying regulators of RAD51, we identified macrophage erythroblast attacher (MAEA), a core component of C-terminal to Lish (CTLH) E3 ubiquitin ligase complex, as a regulator of the HR pathway. Loss of MAEA impairs RAD51 recruitment at stalled RFs, leading to increased sensitivity to RS-inducing agents and excessive degradation of nascent DNA strands. Mechanistically, MAEA associates with and mediates the ubiquitylation of Ku80, enabling its removal from RF ends and facilitating the loading of RAD51. Notably, MAEA deficiency is associated with a developmental disorder involving microcephaly, craniofacial abnormalities, ocular defects, and heart malformations. Functional assays show that disease-linked MAEA variants (R34C, E349G, Y394D, and M396R) are defective in RS response. These findings establish MAEA as an essential factor in RF protection and genome integrity.

Humans↗
PubMed2025

Innate immune sensing and signaling: Co-opted for genome surveillance? Implications for tumorigenesis.

Innate immune signaling is traditionally associated with the response to pathogenic infection. However, emerging evidence suggests that nuclear innate immune sensors and their downstream pathways may also serve as a critical mechanism for genome surveillance. This review explores a model in which DNA sensors such as mouse IFI204 and IFI205 (IFI16 in humans) localize to replication forks, where they detect endogenous aberrant DNA structures and initiate an interferon-stimulated gene (ISG) transcriptional program. A key output of this transcriptional program is ISG15, which we find conjugated to fork-associated proteins and facilitates recruitment of the replication fork protection complex, thereby stabilizing replication forks under physiological conditions. We discuss how nuclear innate immune sensors mediate replication stress sensing and examine the broad consequences of downstream ISG transcription across diverse contexts-including its impact on genome stability and its dual roles in modulating tumor cell behavior and the tumor microenvironment. These findings suggest that the innate immune system, through its nuclear DNA sensing arm, may be evolutionarily co-opted for genome surveillance and may influence tumor initiation and therapy resistance. Understanding how innate immune signaling intersects with replication stress could offer mechanistic insights into tumor development and reveal novel therapeutic targets.

Humans↗
PubMed2019

Homologous recombination defects and how they affect replication fork maintenance.

Homologous recombination (HR) repairs DNA double strand breaks (DSBs) and stabilizes replication forks (RFs). RAD51 is the recombinase for the HR pathway. To preserve genomic integrity, RAD51 forms a filament on the 3' end of a DSB and on a single-stranded DNA (ssDNA) gap. But unregulated HR results in undesirable chromosomal rearrangements. This review describes the multiple mechanisms that regulate HR with a focus on those mechanisms that promote and contain RAD51 filaments to limit chromosomal rearrangements. If any of these pathways break down and HR becomes unregulated then disease, primarily cancer, can result.

RAD51 filaments↗
PubMed2024

Reversion from basal histone H4 hypoacetylation at the replication fork increases DNA damage in FANCA deficient cells.

The FA/BRCA pathway safeguards DNA replication by repairing interstrand crosslinks (ICL) and maintaining replication fork stability. Chromatin structure, which is in part regulated by histones posttranslational modifications (PTMs), has a role in maintaining genomic integrity through stabilization of the DNA replication fork and promotion of DNA repair. An appropriate balance of PTMs, especially acetylation of histones H4 in nascent chromatin, is required to preserve a stable DNA replication fork. To evaluate the acetylation status of histone H4 at the replication fork of FANCA deficient cells, we compared histone acetylation status at the DNA replication fork of isogenic FANCA deficient and FANCA proficient cell lines by using accelerated native immunoprecipitation of nascent DNA (aniPOND) and in situ protein interactions in the replication fork (SIRF) assays. We found basal hypoacetylation of multiple residues of histone H4 in FA replication forks, together with increased levels of Histone Deacetylase 1 (HDAC1). Interestingly, high-dose short-term treatment with mitomycin C (MMC) had no effect over H4 acetylation abundance at the replication fork. However, chemical inhibition of histone deacetylases (HDAC) with Suberoylanilide hydroxamic acid (SAHA) induced acetylation of the FANCA deficient DNA replication forks to levels comparable to their isogenic control counterparts. This forced permanence of acetylation impacted FA cells homeostasis by inducing DNA damage and promoting G2 cell cycle arrest. Altogether, this caused reduced RAD51 foci formation and increased markers of replication stress, including phospho-RPA-S33. Hypoacetylation of the FANCA deficient replication fork, is part of the cellular phenotype, the perturbation of this feature by agents that prevent deacetylation, such as SAHA, have a deleterious effect over the delicate equilibrium they have reached to perdure despite a defective FA/BRCA pathway.

Histones↗
PubMed2026

STN1 upregulation promotes PARPi resistance in BRCA2-deficient cancer cells via replication fork protection and suppression of ssDNA gap formation.

PARPi are effective therapy for BRCA1/2 mutant cancers, yet recurrent PARPi resistance frequently develops. The underlying mechanism of PARPi resistance remains largely unresolved. Here, we identify STN1, a component of the CTC1/STN1/TEN1 (CST) complex, as a modulator of PARPi resistance in BRCA2-deficient cells. RNA-seq analysis of PARPi-resistant cancer cells from BRCA2-mutated backgrounds shows largely distinct transcriptomic profiles with limited overlap, suggesting multiple routes to resistance. Notably, STN1 is consistently upregulated in resistant cells. We observe that overexpression of STN1 enhances Olaparib resistance in multiple BRCA2-deficient cell lines and alleviates DNA damage under replication stress. Mechanistically, we find that STN1 overexpression increases RAD51 loading to stalled replication forks while restricting MRE11 recruitment in BRCA2-deficient cells, thereby protecting stalled forks from nascent-strand degradation. Furthermore, STN1 overexpression rescues the accumulation of ssDNA gaps, a major determinant of PARPi sensitivity in BRCA2-deficient cells. Taken together, these findings suggest that elevated STN1 levels can partially compensate for BRCA2 loss by stabilizing stalled replication forks and limiting ssDNA gap accumulation. Our study uncovers a STN1-dependent pathway of replication stress tolerance that promotes PARPi resistance independently of homologous recombination restoration, highlighting STN1 as a potential biomarker and mechanistic contributor to therapeutic resistance in BRCA2-mutated cancers.

PARPi resistance↗
PubMed2024

RNF4 sustains Myc-driven tumorigenesis by facilitating DNA replication.

The mammalian SUMO-targeted E3 ubiquitin ligase Rnf4 has been reported to act as a regulator of DNA repair, but the importance of RNF4 as a tumor suppressor has not been tested. Using a conditional-knockout mouse model, we deleted Rnf4 in the B cell lineage to test the importance of RNF4 for growth of somatic cells. Although Rnf4-conditional-knockout B cells exhibited substantial genomic instability, Rnf4 deletion caused no increase in tumor susceptibility. In contrast, Rnf4 deletion extended the healthy lifespan of mice expressing an oncogenic c-myc transgene. Rnf4 activity is essential for normal DNA replication, and in its absence, there was a failure in ATR-CHK1 signaling of replication stress. Factors that normally mediate replication fork stability, including members of the Fanconi anemia gene family and the helicases PIF1 and RECQL5, showed reduced accumulation at replication forks in the absence of RNF4. RNF4 deficiency also resulted in an accumulation of hyper-SUMOylated proteins in chromatin, including members of the SMC5/6 complex, which contributes to replication failure by a mechanism dependent on RAD51. These findings indicate that RNF4, which shows increased expression in multiple human tumor types, is a potential target for anticancer therapy, especially in tumors expressing c-myc.

Animals↗
PubMed2026

RADX protects against intestinal inflammation by restraining IFI16-mediated innate immunity.

Genomic instability is increased in patients with inflammatory bowel disease (IBD), yet whether it contributes directly to disease pathogenesis remains unclear. Here, we identify RADX, a structural antagonist to RAD51 and a key regulator of replication fork stability, as a critical suppressor of intestinal inflammation by limiting innate immune sensing of replication-associated DNA damage. RADX deficiency exacerbates experimental colitis, with macrophages serving as the principal mediators of this phenotype. Mechanistically, RADX competes with the DNA sensor IFI16 for binding to single-stranded DNA (ssDNA). Loss of RADX promotes ssDNA accumulation, triggering IFI16-dependent activation of NF-κB signaling and inflammasome assembly, thereby driving intestinal inflammation. Consistent with these findings, two RADX variants identified in patients with IBD associate with reduced RADX protein expression, increased DNA damage signaling, and elevated IL-1β levels. Pharmacological inhibition of RAD51 with RI-1 alleviated colitis in both wild-type and Radx-deficient mice. Together, these findings establish a mechanistic link between genome instability and intestinal inflammation, identify a RADX-IFI16 checkpoint that restrains pathogenic innate immune activation, and nominate modulation of replication stress as a therapeutic strategy for IBD.

Animals↗
PubMed2025

The DNA replication checkpoint limits Okazaki fragment accumulation to protect and restart stalled forks.

Understanding how DNA replication forks stall and restart and how the DNA replication checkpoint prevents irreversible fork collapse in molecular detail are crucial for understanding how cells maintain stable genomes and how they prevent the genetic instability that drives cancer. Here, we describe the reconstitution of fork stalling and restart with purified budding yeast proteins. After nucleotide depletion, leading-strand DNA synthesis quickly stops but CMG helicase continues to unwind, and Okazaki fragments continue to initiate on the lagging strand. Incomplete Okazaki fragments sequester PCNA, RFC, and DNA polymerases δ and ε, which prevents normal DNA synthesis restart and exposes nascent DNA to nuclease attack. The DNA replication checkpoint restrains fork progression, which limits this sequestration, protecting stalled forks from collapse and ensuring restart.

DNA Replication↗
PubMed2026

Cohesin reshapes replication fork contacts to aid fork slowing and reversal.

DNA replication forks can be challenged by cancer chemotherapeutic treatments, leading to accumulation of single-stranded DNA and slowdown of DNA synthesis. The marked plasticity of replication forks under replication stress ensures fork stability, damage tolerance and complete genome duplication1. Initiation and progression of replication forks occur in a three-dimensionally organized genome. DNA loop extrusion by the cohesin complex organizes the genome2 and regulates the initiation and positioning of DNA replication origins3,4. Although transient interaction of sister forks was recently reported during unperturbed replication5, the functional relevance of fork contacts during replication stress and the role of cohesin in this context remain unknown. Here we show that cohesin-mediated loop extrusion rearranges nascent DNA contacts at stressed replication forks to promote genome stability. Using auxin-inducible degron6, separation-of-function mutants7-9 and a newly developed Micro-C-based technique to capture chromatin contacts at nascent DNA (Repli-C), we found that loop-extruding cohesin accumulates at stalled replication forks, limiting sister-fork coupling in favour of inter-replicon contacts. This process promotes active fork slowing and reversal by preventing PRIMPOL action on single-stranded DNA1. These findings show that the replication stress response is not merely an accumulation of individual regulatory events, but is topologically integrated across the genome through cohesin loop extrusion. While providing a new function for loop-extruding cohesin, our results indicate the potential impact on cancer therapy of frequent cohesin mutations in tumours10.

Journal Article↗
PubMed2016

A mechanism for 1,4-Benzoquinone-induced genotoxicity.

Benzene is a common environmental toxin and its metabolite, 1-4-Benzoquinone (BQ) causes hematopoietic cancers like myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). BQ has not been comprehensively assessed for its impact on genome maintenance, limiting our understanding of the true health risks associated with benzene exposure and our ability to identify people with increased sensitivity to this genotoxin. Here we analyze the impact BQ exposure has on wild type and DNA repair-defective mouse embryonic stem (ES) cells and wild type human cells. We find that double strand break (DSB) repair and replication fork maintenance pathways including homologous recombination (HR) and Fanconi anemia (FA) suppress BQ toxicity. BQ-induced damage efficiently stalls replication forks, yet poorly induces ATR/DNA-PKCS responses. Furthermore, the pattern of BQ-induced γH2AX and 53BP1foci is consistent with the formation of poly(ADP-ribose) polymerase 1 (PARP1)-stabilized regressed replication forks. At a biochemical level, BQ inhibited topoisomerase 1 (topo1)-mediated DNA ligation and nicking in vitro; thus providing mechanism for the cellular phenotype. These data are consistent with a model that proposes BQ interferes with type I topoisomerase's ability to maintain replication fork restart and progression leading to chromosomal instability that has the potential to cause hematopoietic cancers like MDS and AML.

Animals↗
PubMed2025

Nanopore-based sequencing of active DNA replication reveals key principles of metazoan replication fork progression, origin and termination sites.

Balancing replication fork progression and origin usage is essential to maintain genome stability, but measuring replication fork progression rates and origin usage throughout the genome has been challenging. Here, we use nanopore sequencing combined with DNAscent to measure replication fork progression together with origin and termination site usage with single-molecule precision throughout the Drosophila genome with nearly full genome coverage. We find that replication fork progression rates are not uniform throughout the genome. Rather, fork progression is slowest in euchromatin, and this is not correlated with active transcription. Replication origins are also influenced by chromatin, but the exact position of initiation is highly variable and are often several kilobases away from ORC binding sites. Termination sites lack any chromatin or sequence motifs and appear nearly random throughout the genome. By measuring DNA replication dynamics at near full genome coverage, our work reveals key principles of metazoan replication dynamics.

Journal Article↗
PubMed2026

Distinct functions of mammalian RAD51 paralogs in genome maintenance.

RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) are evolutionarily conserved essential proteins for cell survival and genome maintenance. RAD51 paralogs were originally identified to play a role in homologous recombination-mediated repair of DNA double-strand breaks (DSBs). However, investigations over the last decade have uncovered new roles of RAD51 paralogs beyond DSB repair in replication stress responses, including replication fork progression, fork stability, and its restart. Recent structural studies have not only uncovered the molecular architecture of previously known RAD51 paralog complexes but also identified novel paralog complex assemblies, providing mechanistic insights into their various genome-maintenance functions. Additionally, a role for RAD51 paralogs in resolving R-loops has been identified, and studies with cancer-associated variants suggest that RAD51 paralogs are potential determinants of cancer susceptibility and therapeutic responses. In the present review, we highlight the recently deciphered structures and novel functions of RAD51 paralog complexes and discuss the clinical and therapeutic implications.

Rad51 Recombinase↗
PubMed2026

Linker Histone H1 Phosphorylation Promotes DNA Damage Repair during Replication Stress.

DNA replication fidelity depends on the integrity of the replication fork to prevent DNA damage and preserve genome stability. Disruptions to this process can trigger replication stress, leading to the accumulation of single-strand DNA (ssDNA) and double-strand breaks (DSBs), which drive mutagenesis and ultimately contribute to disease. While the roles of core histones and their post-translational modifications in this context have been more well-studied, far less is known about how linker histones regulate the replication stress response. Here, we demonstrate that the S-phase-phosphorylated form of the linker histone H1 (pH1) plays a key role in DNA damage repair at collapsed replication forks, both in vitro and in cells. Using phosphomimic and phosphonull H1 mutants, we show that phosphorylation enhances H1 assembly with ssDNA. Utilizing intein chemistry for the site-specific incorporation of a photocrosslinker to the C-terminus of H1, we map the direct interactors of H1. We identify phosphorylated H1 at replication forks, where it engages replication machinery and DNA damage response factors, including Histone PARylation Factor 1 (HPF1). We further demonstrate that ssDNA induces pH1-HPF1 interactions that promote liquid-like assemblies, correlating with reduced DNA damage and histone PARylation. Consistent with this role, reduction of total H1 increases cellular sensitivity to DNA damage, a phenotype that is partially rescued by reintroduction of H1.4. Together, these findings establish pH1 as a new regulator of DNA damage repair at collapsed replication forks through the controlled sequestration of repair factors.

Journal Article↗
PubMed1992

Energy flow considerations and thermal fluctuational opening of DNA base pairs at a replicating fork: unwinding consistent with observed replication rates.

The effect of an open loop of various sizes on the thermal stability of the adjoining intact base pairs in a duplex DNA chain is studied in a lattice model of Poly(dG).Poly(dC). We find that for a Y-shaped fork configuration the thermal fluctuation at the fork is so enhanced that the life time of the adjoining base pair is much smaller than the 1 millisecond time scale associated with helicase separation of a base pair in some systems. Our analysis indicates that thermal fluctuational base pair opening may be of importance in facilitating the enzyme unwinding process during chain elongation of a replicating DNA. It is most likely that the thermal fluctuational opening of the base pair at the junction of a replicating fork is fast enough so that a DNA unwinding enzyme can encounter an unstacked base pair with reasonable probability. This conclusion can explain several experimental observations regarding the temporal relationship between ATP hydrolysis by accessory proteins and primer elongation by a holoenzyme complex in ssDNA. We also discuss a mechanism by which the energy associated with ATP hydrolysis may enhance the thermal driven base opening mechanism.

Adenosine Triphosphate↗
PubMed2025

Enzymatic and Structural Roles of Candida albicans Rev1 in DNA Damage Response and Disseminated Candidiasis.

Translesion DNA synthesis (TLS) is a fundamental biological process that enables DNA replication through various lesions to ensure genome stability and to prevent cell death due to replication fork collapse. Rev1, a member of Y-family DNA polymerase (Pol), functions in concert with a B-family enzyme Polζ in promoting TLS through various lesions. Interestingly, for such a function, the catalytic activity of Rev1 seems to be dispensable in Saccharomyces cerevisiae. Unlike Polζ, which possesses robust DNA polymerase activity, biochemical assays suggest that Rev1 predominantly incorporates a "C" opposite any templating residues, but the biological relevance of this activity of Rev1 remains elusive. Here we characterized Rev1 from Candida albicans, an opportunistic fungal pathogen responsible for maximum casualties due to systemic candidiasis in immunosuppressed individuals. Concerted genetic analyses of several Rev1 mutants in various DNA-damaging conditions suggested that in most lesion bypasses except 4-NQO-induced DNA lesions, the catalytic role of Rev1 is not important. However, simultaneous interactions of BRCT and the C-terminal domain of Rev1 with PCNA and Polζ, respectively, enable Rev1 to be essential during TLS. DNA damage recovery and mutagenesis assays further confirmed the lesion-specific roles of various domains of Rev1. Contrary to ex vivo data, animal studies suggested that CaRev1 is dispensable for systemic candidiasis development. We discuss the possible involvement of other TLS DNA polymerases in DNA damage response while C. albicans replicates and establishes itself in the host.

Candida albicans↗
PubMed2026

Single-molecule tracking of RNA-DNA hybrid removal enzymes important for lagging-strand replication.

The formation of RNA-DNA hybrid (RDH) primers by primase is an essential step in the recruitment of DNA polymerase during replication initiation and for the synthesis of each Okazaki fragment on the lagging strand. In addition to primers, RDHs form through misincorporation of ribonucleotides by DNA polymerase during elongation and by formation of R loops during transcription. R loops are three-stranded structures that form when the nascent mRNA anneals to the template DNA strand, displacing the complementary DNA strand. The persistence of RDHs is deleterious to genome stability in all cells because they increase susceptibility to mutations, impaired replication fork progression, DNA double-stranded breaks, and genomic rearrangements. In many bacteria, it is well established that components of the replicative DNA polymerase form a macromolecular complex that can be imaged using single-molecule or ensemble fluorescence approaches. The spatiotemporal regulation of proteins involved in RDH removal during lagging-strand maturation is less clear. Here, we study three proteins that are involved in the removal of RDHs from the lagging strand during DNA replication in the Gram-positive bacterium Bacillus subtilis: DNA polymerase I (Pol I), FenA, and RNase HIII. We characterized the behavior of each PAmCherry-tagged lagging-strand enzyme in living cells using single-particle tracking photoactivated localization microscopy. We find that all three proteins are highly mobile, suggesting residence times at their target substrates are below our temporal resolution. We also find evidence that Pol I activity is modulated through interaction with the replisome, whereas FenA and RNase HIII are regulated through access to the nucleoid. Our results provide new insight into how enzymes are recruited to resolve RDHs during lagging-strand replication in vivo.

DNA Replication↗